The long-term objectives of this proposal are to develop the means whereby human hematopoietic stem cells (HSC) can be manipulated in culture to provide a cellular source for bone marrow transplantation and target cells for gene transfer. The working hypothesis is that specific hematopoietic growth factors (HGFs) can be identified that are crucial to the regulation of stem cell self-renewal and differentiation. This hypothesis will be tested in two ways: first, (Aim 1) murine, simian, and human stem and progenitor cells will be assessed for their self- renewal and differentiation capacity after incubation in selected HGF combinations; and second (Aim 2), the genes for candidate HGF receptors will be disrupted in embryo-derived stem cells (ES) to provide a critical measure of their importance in vivo. In Aim 1, species-appropriate methods will be used to purify or enrich for hematopoietic stem cells and separate them from committed progenitors. These cells will be incubated in a combination of HGFs for 6-7 days and then evaluated for their stem and progenitor cell and T lymphocyte content, both in vitro and in vivo in murine and simian species, and in vitro in the human studies. In addition, the effect of combined HGFs on the efficiency of gene transfer into stem and progenitor cells and the number of clones that contribute to hematopoiesis will be determined. In Aim 2, genes for the interleukin-3 receptor will be disrupted in ES cells by homologous recombination and evaluated for function in vitro and in vivo. Both the alpha and beta subunits of the IL-3 R will be disrupted using a vector that allows positive-negative selection. ES cells with heterozygous mutations of IL-3 Ralpha or beta will be injected into blastocysts and transferred to foster mothers. Mice with germ line integration will be bred to homozygosity and interbred to yield IL-3 R alpha, beta, and alphabeta "null" animals. In addition, the heterozygous mutant ES cells will be "homozygosed" in vitro and then evaluated directly in chimeric animals by glucose phosphate isomerase analysis. Last, we plan to use IL-3 R "null" murine ES cells for structure function analysis of the human IL-3 R/GM-CSF R components. In these ways, we hope to identify methods to expand the population of pluripotent HSC for eventual clinical use in bone marrow transplantation, either unmodified or, ultimately, modified by gene insertion. In addition, we hope to establish gene mutation methods that would provide a powerful means to investigate the physiological importance of IL-3 and other HGFs in hematopoiesis.